Various thrombocytopoietic disorders can cause life-threatening hemorrhage. There have been intense efforts in developing thrombopoietic growth factors. The primary cytokine for megakaryocyte and platelet development is thrombopoietin (Tpo). Tpo, signaling through its receptor Mpl, also plays important roles in hematopoietic stem/progenitor cell (HSPC) expansion. Tpo binding to Mpl induces a conformational change in the Mpl/ Janus kinase 2 (JAK2) complex, which leads to the activation of JAK2 and triggers a cascade of signaling events. The FDA recently approved two thrombopoiesis- stimulating agents: AMG531 (Romiplostim), a Tpo peptide mimetic, and Eltrombopag (SB497115), a small molecule Mpl agonist. We show in collaborative studies that Eltrombopag- family compound, SB559457, stimulate human bone marrow (BM) megakaryopoieis. Strikingly, SB559457 and Eltrombopag, which bind to the transmembrane (TM) domain of human Mpl, robustly activate MAPK and mTOR/S6K pathways, but only minimally activate Stat5. The 1-helical conformation of the receptor TM and juxtamembrane (JM) is critical for the proper activation of JAK2 and downstream signaling cascade. We therefore hypothesize that the Mpl/JAK2 complex adopts different orientation/ conformations when stimulated by Tpo/AMG531 or Eltrombopag. Eltrombopag ligation results in a suboptimal Mpl conformation that partially activates JAK2/Stat5 pathway, which may result in different cellular outcomes in megakaryocytes and HSPCs. Specific Aim 1: Dissect orientation- dependent Mpl signaling induced by Tpo, AMG531, and Eltrombopag in cell lines. Attempts to solve the structure of Mpl intracellular domain have failed, we therefore will introduce alanine insertion mutations to the Mpl TM/JM helix to dissect the orientation-dependent Mpl signaling induced by Tpo, AMG or Eltrombopag. Specific Aim 2: Dissect signaling pathways induced by Tpo, AMG531, and Eltrombopag in primary BM megakaryocyte culture. After identifying the Mpl mutants that differentially affect Tpo, AMG531 versus Eltrombopag function in cell lines, we will functionally validate the cell proliferative effects and signaling pathways induced by different Mpl agonists in primary BM cultures. Specific Aim 3: Dissect orientation- dependent signaling and hematopoiesis induced by Tpo, AMG531, and Eltrombopag in BM reconstituted mouse models expressing human Mpl. We will test the results gained from last aims in physiological settings. We will administrate Tpo, AMG531, or Eltrombopag to mice that are reconstituted with wild type or mutant human Mpl, and compare their abilities to support megakaryopoiesis and HSPC expansion, as well as the long-term efficacy and safety profiles. The research proposed here aims to achieve a mechanistic understanding of how different classes of thrombopoietic growth factors activate Mpl/JAK2- mediated signaling, and regulate megakaryocytes and HSPC expansion. These studies could potentially lead to better therapeutic strategies in utilizing different thrombopoietic promoting drugs in the treatment of autoimmune and iatrogenic thrombocytopenia. PUBLIC HEALTH RELEVANCE: Various thrombocytopoietic disorders, such as idiopathic thrombopoietic purpura and chemotherapy- induced thrombocytopenia in cancer patients, can cause life-threatening bleeding, thereby imposing great risk to the patients. The research proposed here aims to achieve a mechanistic understanding of how different classes of thrombopoietic growth factors elicit signaling transduction and regulate multiple aspects of hematopoiesis. Since the efficacy and safety of the long-term therapy with these drugs rely on their action on both lineage committed megakaryocytes and multi-potential stem/progenitor cells, these studies could potentially lead to better therapeutic strategies in utilizing different thrombopoietic promoting drugs in the treatment of autoimmune and iatrogenic thrombocytopenia.